The present invention is in the area of biotechnology, in particular the preparation of viable products by fermentation of microorganisms able to form the viable products of interest. This includes for example the preparation of low molecular weight compounds, for instance of dietary supplements or pharmaceutically relevant compounds, or of proteins for which, because of their diversity, there is in turn a large area of industrial uses. In the first case, the metabolic properties of the relevant microorganisms are utilized and/or modified to prepare the viable products; in the second case, cells which express the genes of the proteins of interest are employed. Thus in both cases, genetically modified organisms (GMO) are mostly involved.
There is an extensive prior art on the fermentation of microorganisms, especially also on the industrial scale; it extends from the optimization of the relevant strains in relation to the formation rate and the nutrient utilization via the technical design of the fermenters and up to the isolation of the valuable products from the relevant cells themselves and/or the fermentation medium. Both genetic and microbiological, and process engineering and biochemical approaches are applied thereto. The aim of the present invention is to improve this process in relation to a common property of the microorganisms employed, which impairs the actual fermentation step, specifically at the level of the genetic properties of the strains employed.
For industrial biotechnological production, the relevant microorganisms are cultured in fermenters which are configured appropriate for their metabolic properties. During the culturing, they metabolize the substrate offered and, besides the actual product, normally form a large number of other substances in which there is ordinarily no interest and/or which—as explained hereinafter—may lead to difficulties in the fermentation or the working up.
Fermentations are normally very complicated processes in which a large number of different parameters must be adjusted and monitored. Thus, for example, aerobic processes are very often involved, meaning that the microorganisms employed must be supplied adequately with oxygen throughout the fermentation (control of the aeration rate). Further examples of such parameters are the reactor geometry, the continuously changing composition of the nutrient medium, the pH or the CO2 formation rate. A particularly important parameter both in terms of the economics and in relation to the process management per se is the necessary energy input, for example via agitation systems which ensure that the reactor content is mixed as thoroughly as possible. In addition, besides the substrate distribution, also an adequate supply of oxygen to the organisms is ensured.
After completion of the fermentation it is normally necessary, besides the removal of the producer organisms, for the valuable product of interest to be purified and/or concentrated from the so-called fermenter slurry. The working up process can include for example various chromatographic and/or filtration steps. Thus, besides the content of valuable products, also decisive for the success of the overall working up process are the biophysical properties of the fermenter slurry, especially its viscosity immediately after completion of the fermentation.
The properties thereof are also influenced by the metabolic activities of the chosen microorganisms, it also being possible for unwanted effects to occur. These include for example a frequent increase in the viscosity of the nutrient medium during the fermentation. This impairs the mixing and thus the transport of matter and the oxygen supply inside the reactor. Additional difficulties mostly arise during the subsequent working up because increased viscosities considerably impair for example the efficiency of filtration processes.
It is known in particular that species of the genus Bacillus produce slime which consists essentially of poly-gamma-glutamate (PGA) and/or -aspartate, meaning polyamino acids linked via the relevant gamma peptide bonds. In scientific studies on Bacillus subtilis it is mainly the three genes ywsC, ywtA and ywtB and the gene products derived therefrom which are connected with the production of poly-gamma-glutamate; the gene product of ywtD is involved in the degradation. The general designation “ywt” for genes is in this connection synonymous with the abbreviations “cap” and “pgs” which are in common use for the same functions. This is explained below.
The publication “Physiological and biochemical characteristics of poly gamma-glutamate synthetase complex of Bacillus subtilis” (2001) by M. Ashiuchi et al., in Eur. J. Biochem., volume 268, pages 5321-5328, describes the PgsBCA (poly-gamma-glutamate synthetase complex BCA) enzyme complex, which consists of the three subunits PgsB, PgsC and PgsA, from B. subtilis. This complex is, according to this, an atypical amide ligase which converts both the D and the L enantiomer of glutamate into the corresponding polymer. According to this publication, a gene disruption experiment described therein is to be regarded as proof that this complex is the only one catalyzing this reaction in B. subtilis. 
Y. Urushibata et al. demonstrate in the publication “Characterization of the Bacillus subtilis ywsC gene, involved in gamma-polyglutamic acid production” (2002), in J. Bacteriol., volume 184, pages 337-343, inter alia via deletion mutations in the three genes ywsC, ywtA and ywtB, that the three gene products responsible in B. subtilis for the formation of PGA are encoded by these three genes. They form in this sequence and together with the subsequent gene ywtC a coherent operon in this microorganism.
The fact that a further gene relevant for the metabolism of PGA is located in the genome of B. subtilis downstream from ywtC in its own operon is shown by T. Suzuki and Y. Tahara in the publication “Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation” (2003), J. Bacteriol., volume 185, pages 2379-2382. This gene codes for a DL-endopeptidase which is able to hydrolyze PGA and thus can be referred to as gamma-DL-glutamyl hydrolase.
An up-to-date survey of these enzymes is additionally provided by the article “Biochemistry and molecular genetics of poly-gamma-glutamate synthesis” by M. Ashiuchi and H. Misono in Appl. Microbiol. Biotechnol., volume 59, pages 9-14 of 2002. The genes homologous to pgsB, pgsC and pgsA and coding for the PGA synthase complex in B. anthracis are referred to therein as capB, capC and capA. The gene located downstream is referred to according to this article as dep (for “D-PGA depolymerase”) in B. anthracis and as pgdS (for “PGA depolymerase”) in B. subtilis. 
In the current state of the art, these enzymic activities are already in positive use mainly for preparing poly-gamma-glutamate as raw material, for example for use in cosmetics, although their exact DNA sequences and amino acid sequences have not to date been known—especially from B. licheniformis. Thus, for example, the application JP 08308590 A discloses the preparation of PGA by fermentation of the PGA-producing strains itself, namely of Bacillus species such as B. subtilis and B. licheniformis; the isolation of this raw material from the culture medium is also described therein. B. subtilis var. chunkookjang represents, according to the application WO 02/055671 A1, a microorganism which is particularly suitable therefor.
Thus, in some fermentations there is an interest in GLA as the valuable product to be produced by the fermentation.
However, the interest in all other fermentations is to prepare other valuable products; in this connection, the formation of polyamino acids means, for the reasons stated above, a negative side effect. A typical procedure for mastering the increased viscosity of the fermentation medium attributable to the formation thereof is to increase the agitator speed. However, this has an effect on the energy input. In addition, the fermented microorganisms are exposed thereby to increasing shear forces representing a considerable stress factor for them. In the end, very high viscosities cannot be overcome even thereby, so that premature termination of the fermentation may be necessary, although production could otherwise be continued.
Slime formation, as a negative side effect of numerous fermentation processes, may thus have negative effects on the overall result of fermentation for diverse reasons. Conventional methods for successfully continuing fermentations in progress despite an increasing viscosity of the nutrient medium can be designated only as inadequate, especially because they do not represent a causal control.